Micromethod and device for rapid detection, enumeration and identification of entities

ABSTRACT

This invention describes a method and device for rapid detection, enumeration and identification of microorganisms in liquid or air samples without preliminary growth. It based on the production and accumulation of absorbent or fluorescent molecules during reactions between artificial substrates and enzymes in the super small channels of the sampling-detecting unit, which is a part of sample treating device. The enzymes of cells or enzymes attached to the cell body through antibody-enzyme conjugate produce easily detectable concentration of colored or fluorescent molecules in small volume much faster than in a large volume. Channels contain cells look like colored or fluorescent dots under light or fluorescent microscope.

BACKGROUND OF THE INVENTION

[0001] Modern methods for detection, enumeration and identification of microorganisms are based on two main trends. The first, provide analysis after preliminary growth on special nutrient media. The second trend doesn't need preliminary growth. The first trend comprises big amount of different chemical, biochemical, physical, optical and other methods, but needs many hours or days for preliminary growth to produce enough homogeneous cells or colonies. The second trend comprises methods of Microscopy, Flow Cytometry and PCR. They allow analyzing of cells immediately after sampling and sample treatment. Analyzing of single cells without preliminary growth belongs to rapid micromethods.

[0002] Microscopy (light and fluorescent, visual or automated) and Flow Cytometry (absorbent, fluorescent or scattering) needs a treatment of cell body by an absorbent or fluorescent dyes. Utilization of antibodies with attached fluorescent molecules helps in rapid identification of single cells. Higher concentrations of colored molecules increases the reliability of analysis.

[0003] One of the well known markers for detection and identification of cells are artificial substrates—non colored or fluorescent substances cleaved by enzymes or enzymatic groups with the production of light absorbent or fluorescent molecules. Artificial substrates are broadly used for detection of live microorganisms, detection by unique enzyme, identification by enzymatic profiles or utilizing in Enzyme-Immunogical Analysis (EIA). Some of the artificial substrates produced non-soluble in cell body precipitates (Tetrazolium salts, Fluorescein based substrates, Rezorufin and other). This feature is useful for Microscopy and Flow Cytometry, because of specifically colored cell bodies. Other substrates produce soluble derivatives that leak in outer space and colored all volume (4-Methylumbelliferone, Tetramethylbenzidine, Fluorescein in alkaline environment and other). This group of artificial substrates could produce large amounts of absorbent or fluorescent molecules because they don't accumulated in cells and don't oppress biochemical pathways of living cells as precipitates do.

[0004] Retention of fluorescent or absorbent molecules leaking out cells or produced in EIA in a small space around a single cell could create easily detectable concentration of these molecules. Utilization of useful features of soluble absorbent or fluorescent molecules together with inventing a simple hand-held device for cell sampling and their immediately treating for detection or identification purposes is the subject of the present invention.

[0005] Simple and rapid detection, enumeration and identification of single prokaryotic or eukariotic cells is very important for medical microbiology, cytology, environmental science, finding of pollutants microorganisms in food and pharmaceutical industry, epydemiology, public and military defense, scientific research and other areas.

SUMMARY OF INVENTION

[0006] The present invention is based on the production and accumulation of absorbent or fluorescent molecules during reactions between artificial substrates (AS) and enzymes in the super small channels of the special sampling-detecting unit (SDU). SDU contains a multitude of super small channels and a membrane filter on a solid base for retention of single particles. Sampling—filtration—from air or liquid. SDU is a part of a hand-held sampling device, which looks like a cylinder with a forcer. It allows the trapping of cells in channels and pass reagents and the washing fluids only in one direction: from the upper side to the lower side. The cells, if any in a sample, pass channels during sampling and are trapped on the filter surface. After this solution containing AS is added to the channels. AS reacts with the enzymes of live cells (detection of live cells) or special indicator enzymes or enzymes previously attached to cells through antibody (EIA version for single cell-single cell identification). After a short incubation period—from minutes to tens minutes—channel containing cell looks like bright fluorescent or colored dot. Therefore, detectable concentration of fluorescent or colored molecules reached only as a result of accumulation in a very small volume—greater than the volume of a single cell only in several thousands—tens thousand times. FIG. 2 shows enlarged channels where one of them contains cell and colored molecules (FIG. 2; 1—upper orifice of the channel, 2—channel with cell and colored molecules, 3—filter for trapping of the cells). Optical object greater than cell in thousands-tens thousand times could be easily detected with modern light or fluorescent optics. The positive effect of accumulation of the products of colored reactions in very small volume could be illustrated by the following calculations:

[0007] Lets divide one milliliter (10¹² μk³) of liquid containing 25·10⁶ cells on smaller parts.

[0008] Each 0.2 ml (2·10¹¹ μk³) (the well of 96-well plate) will contain 5·10⁶ cells. One hundred cells will be in a volume 4·10⁶ μk³. Volume 4·10⁴ μk³ will contain only one cell. This volume corresponds to channel of SDU with dimensions: diameter of channel=10 μk and length of channel=500μk. All these volumes with cells, including the smallest, corresponding to the concentration 25·10⁶ cells/ml. This high concentration of cells can produce easily detectable concentration of colored or fluorescent molecules from enzyme-artificial substrate reactions. It means that one cell in 4·10⁴ μk³ (one channel of SDU) will produce the same easily detectable concentration of the same molecules in the same time as 25·10⁶ cells in one ml or 5·10⁶ cells in 0.2 ml.

[0009] This invention differs from other rapid micromethods by utilizing sampling-detection unit, which consists of an array of very small channels for retaining the products of enzyme-substrate reactions from one single trapped cell. The processes of sampling and sample treatment cooperated in the same device comprises sampling-detection unit. The results—detection, identification and enumeration of single entities. These results could easily be found with usage of regular fluorescent or light microscope or other simple optical devices for this purpose or automated instruments for microanalysis.

DETAILED DESCRIPTION OF THE INVENTION

[0010] This invention provides a simple device and method for sampling of cells from liquid or air, easy treatment procedures of single trapped cells and retention of colored molecules—products of reactions in very small volumes to reach detectable concentration.

[0011] It is well understandable and currently used in practice that dividing of a sample on many different small parts helps to detect cells concentration faster. This effect depends on faster reaching a detectable concentration in a small volume rather than in a large volume. Thus, U.S. Pat. No. 5,716,798 describes the method for rapid detection of microorganisms in a container divided on a plurality of discrete zones, each of which can be separately monitored for microbial presence by reaching detectable cell concentrations after preliminary growth in some zones. This method gives timesaving of 10 to 40% in comparison with other methods. On the same effect based U.S. Pat. No. 5,770,440. The present invention differs from these patents by the analyzation of a single cell. No time consuming preliminary growth and nutrient media are necessary.

[0012] U.S. Pat. No. 4,959,301 is based on dividing of a sample with a viable biological entities on large amount of microdroplets and detecting entities by growth or by biochemical reactions of a single entity in a droplet. This method can indicate single cell in less than 30 minutes in some variants. Nevertheless, it is technologically complicated. Microdroplets produced with different volume and needs statistical analysis for calculating results. This method could be reproduced only in laboratory by high professional personnel. The present invention has the following advantages:

[0013] super small channels that have equal volume: no statistical analysis is necessary;

[0014] droplets that needs special manipulations against drying, but channels has ratio diameter/length=1/30-1/50 and, therefore, an extremely small square of evaporation. No special actions against drying are necessary;

[0015] the volume of a channel smaller than an average droplet, therefore detectable concentrations could be reached faster.

[0016] The effect of increasing of concentration in small volume used in EIA and ELISA, also. Thus, well-known 96-wells immunological plates now produced with 384 and even 912 wells on the plate of the same size (128×86 mm). A smaller volume gives opportunity to reach detectable concentrations of absorbent or fluorescent molecules faster, or use smaller concentrations of homogeneous cells. Nevertheless, these plates and procedures of identification could be used only for identification of homogeneous cells in concentrations of hundreds-thousands cells per ml.

[0017] Our own experiments and calculations were done with vegetative cells Bacillus cereus. They show that one live cell of Bacillus cereus (TSA, 17 hours at 37° C.) produces around 3,000,000 fluorescent molecules of 4-Methylumbelliferone (MU) from 4-Methilumbelliferyl acetate (MUA) per minute. To find fluorescence by the naked eye in the quartz cell of fluorometer Perkin-Elmer LS-5 with maximum excitation 350 nm. needs concentration of MU around 6·10{circumflex over ( )}15 molecules per ml. This concentration in a volume 0.01 mm³ (10³ μk³) could be produced by one cell of Bac. cereus in 2 minutes. To reach the same concentration in 0.1 mm³ (10{circumflex over ( )}6 μk³) needs 33 hours of one cell incubation. In 1 mm³ (10{circumflex over ( )}9 μk³) needs 3.8 years of incubation. 0.02 cm³ (2·10{circumflex over ( )}10 μk³)—76 years. 0.2 cm³ (the volume of 96-wells well)—760 years. 1 cm³—3800 years of incubation.

[0018] Thus, the volumes for reaction needs to be as small as possible to reach detectable concentration in a real time, but large enough to be detected by simple optical methods. Modern rapid micromethods based on concentration of dye in a cell body (Flow Cytometry, automated microscopy) needs special complicated technique (scanning of surface, special flow stream device) to find single cell on a filter, slide or in a flow stream because of very small size of an object, around 0.5-5 μk³.

[0019] The size of the object (fluorescent or colored channel) according the present invention in thousands-tens thousands larger. Therefore, simple optics with small multiplication could be employed. No special complicated technique to find this big object is needed. Thus, the present invention could be employed in the field studies and by not high level professionals. The price of analysis could be reduced strongly.

[0020] Another important part of this invention is the usage of artificial substrates for different enzymes or enzymatic groups for production of detectable concentrations of absorbent or fluorescent molecules. They are broadly used for detection of enzymatic activities. Many different artificial substrates based on chromogenic molecules: 2-Nitrophenol, 4-Nitrophenol, 5-Bromo-4-chloro-3-indoxol, 3-Indoxol, 5-Bromo-6-chloro3-indoxol, 6-Chloro-3-indoxol, 5-Iodo-3-indoxol, N-Methylindoxol, 3,3′,5,5′-Tetramethylbenzidine dihydrochloride and other. Other artificial substrates based on fluorescent molecules: 4-Methylumbelliferone, 7-Amido-4-methylcoumarin, Fluorescein, Eosine and others. They cover a large spectrum of different enzymes: Glycosidases, Esterases, Phosphatases, Peptidases, Sulfatases, Dehydrogenases and special enzymes like Horseradish-Peroxidase, β-D-galactosidase or a special aminopeptidase.

[0021] Different analytical methods could be produced on their base and device described in the present invention. Thus, detection of a single live microorganism in a sample could be done with usage of artificial substrates for big groups of enzymes always present in any live microorganism. For example 4-Methylumbellyferyl phosphate for Phosphatases or 4-Methylumbellyferyl acetate for Esterases or their mixture. Detection of some important microorganisms could be done with exploration of their unique enzymes. For example, 4-Methylumbelliferyl-β-D-galactopyranoside as reliable indicator of β-D-galactosidase - a unique enzyme of Escherichia coli.

[0022] Identification of a single cell trapped in a channel could be done with an enzyme attached to an antibody, which attached to antigens of an investigated cell: EIA version for a single cell.

[0023] Identification of a single cell, also, could be done by enzymatic profiles. In this case a trapped cell will produce fluorescent product from the first substrate which after the measurement of the amount of fluorescence will be washed out, after that, with a second substrate, third and so on. No special instrument for these purposes exists currently.

[0024] Procedure for sampling and treatment:

[0025] Reactions Enzyme(s)—Artificial substrate(s) are to carry out into the channels of SDU (FIG. 1, the size of channels orifices is much smaller than it shown on the picture; FIG. 2, 2). SDU is a part of the device for sampling, trapping of cells in channels, if any, and treatment procedures. Treatment procedures include: accurate addition of an artificial substrate solutions, antibody-enzyme complex and other reagents, if needs, to each channel and washing solutions for washing out not needful molecules (for example surplus of antibody-enzyme complex or during changing artificial substrates when profile is investigated). All liquids pass device from upper side to lower side, because, passing by in another direction could wash out the trapped cells from the channels.

[0026] The general principle of a hand-held device work is following:

[0027] 1. Liquid sample, possibly containing microorganisms, placed in the chamber of device (FIG. 3, 5).

[0028] 2. Plunger (FIG. 3, 4) press sample through SDU (FIG. 3, 7-10). Cells, if any, trapped in the channels on the surface of filter (FIG. 1, 3; FIG. 2, 3; FIG. 3, 8).

[0029] 3. Cap (FIG. 3, 12) close orifice (FIG. 3, 11). Channel for equalization of air pressure (FIG. 3, 6) inside chamber and outside pressure opened by rotating inner cylinder (FIG. 3, 3) against outer cylinder (FIG. 3, 2). This helps to prevent reverse exit of trapped cells from channels. Channel for pressure equalization could have any other construction. For example closing and opening of channel (FIG. 3, 6) could be done by usage of the spigot.

[0030] 4. Small amounts of artificial substrate(s) added to the chamber of the device and the piston press it into channels of SDU. All channels filled with artificial substrates.

[0031] 5. Incubation several minutes-several tens minutes at room temperature or higher. It depends on analyzing object.

[0032] 6. The device is untwisted and SDU placed under the Microscope (light or fluorescent), scanning microscope or an other eligible optical instrument.

[0033] 7. The channels containing live cells look like colored (absorbent version) or fluorescent dots (FIG. 2). The diameter of the dots 15 μk (length of each channel 500 μk and volume 88000 μk³) in our experiments, but could be more or less. This size is big enough to use a small multiplication of the microscope: ×40-×100. Usage of a small multiplication allows the checking of all SDU surfaces rapidly, in several minutes, and even to detect single live cells and enumerate them. Automatization of this process with microscanning systems could make this process faster.

[0034] In case of identification by EIA for a single cell after step 3 needs to add:

[0035] 3′. A solution of conjugate on the antibody with a marker enzyme added to the chamber (FIG. 3, 1, 5). Mixture incubated needful time to attach conjugate to antigens of entity.

[0036] 3″. Open the channel for equalization of pressure (FIG. 3, 6). Pull the plunger (FIG. 3, 4) up. Fill the device chamber with washing fluid and press the plunger down in order to wash out surplus of conjugate. This procedure could be done more than one time if needs.

[0037] In case of enzymatic profiles steps 4, 5, 6 and 7 could be repeated a number of times. The levels of absorption or fluorescence in the channels need to be measured by a microphotometrical or microfluorimetrical instrument. This method allows identification of many different species in one sample, but it is more longer and complicated than the EIA-method for identification of one sort of cells.

[0038] In case of bioaerosol sampling steps 1, 2 and 3 changed to:

[0039] Bioaerosol sampling could be done after removal of piston from the device and plug negative air pressure into nozzle (FIG. 3, 12).

[0040] Identification of single virus particles seems to be possible by present invention (EIA version for single virus particle). Trapping of single virus particles in SDU could be realized by usage of special filters—membranes (ex. dialysis membrane) or treating of inner walls of channels by specific antibodies, lectines or other reagents for trapping single virus particle in channel or employment of magnetic particles.

EXAMPLE 1 Detection of Live Cells Escherichia coli from Drinking Water

[0041] 100 ml of sample of water pressed through a Device with SDU. 2 ml of fresh solution of 4-Methylumbelliferyl-β-D-glucuronide (0.1 mg/ml) in distilled water pressed through SDU with a black non-fluorescent nitrocellulose filter. All channels filled by the solution of fluorogenic substrate. Incubation 20 minutes at 40° C. After this the incubation device untwisted and the microchannel plate with a filter placed under the fluorescent microscope: ×60, λ excitation 340-380 nm., λ fluorescence 450 nm. Channels containing live E. coli look like blue fluorescent dots. Channels without cells or live cells of other species looks like black dots. The same result by other rapid methods could be reached in 6-8 hours.

EXAMPLE 2 Identification of Brucella melitensis from Milk

[0042] 100 ml of a milk sample previously defatted pressed through Device with SDU with a white nitrocellulose filter. 50 ml of Phosphate buffer pH 8.0 pressed through Device to wash out the rest of the proteins and fat. 2 ml of standard conjugate of antibody for B. melitensis surface antigens and Horseradish Peroxidase added to Device and slowly—part after part, during several minutes—pressed through SDU. After that 50 ml of distilled water pressed through SDU in order to wash out the rest of the conjugate. 2 ml of solution of 3,3′,5,5′-Tetramethylbenzidine added to the Device and pressed through SDU. Incubation 40 minutes at 35° C. After incubation SDU with filter placed under light microscope; ×60. Channels contain B melitensis look like blue dots. Other channels looks like white dots. Even one cell in 100 ml could be found in less than one hour. The regular procedure needs a preliminary growth period of at least 26-28 hours. Flow cytometry needs the same time—tens of minutes, but needs an instrument with a price of around $100,000 and highly qualified personnel. PCR reach the same result in two hours. Complicated technique needs, also. 

What is claimed is:
 1. In a method and device for sampling, detection and/or identification of prokaryotic or eukariotic entities without preliminary growth which method comprises: sampling of entities in multiplicity of super-small hollow and opened from both sides long channels attached or not attached to filtration material for trapping single particles in channel and providing biochemical reactions between enzymes and artificial substrates in order to retent optically active products of these reactions in super-small volume in order to rapidly create detectable concentration of these products.
 2. The method of claim 1 wherein live entities detected by reactions between artificial substrate(s) and enzyme(s) of a live entity.
 3. The method of claim 1 wherein entities identified in reactions between artificial substrate(s) and enzyme(s) precursory attached to an antibody which is connected to antigen of trapped entity.
 4. The method of claim 1 wherein detection or identification monitored by fluorimetric means.
 5. The method of claim 1 wherein detection or identification monitored by colorimetric means.
 6. The device of claim 1 wherein the sample contains or not contains entities pressed through plate contain from multiplicity of long super-small channels on a filter and rigid, but porous plate, which is a part of sampling and sample treating device.
 7. The device for sampling and sample treating of claim 1 wherein special channel could be opened or closed in order to equalize air pressure inside and outside the chamber during piston movement upwards. 